WNT genes encode secreted lipid modified growth factors (Takada et al., 2006; Willert et al., 2003) that exert potent effects on stem cells (Nusse, 2008; Reya and Clevers, 2005; Reya et al., 2003; Willert et al., 2003). Binding of WNT proteins to their cognate receptors encoded by the Frizzled (FZD) gene family initiates various intracellular signaling cascades, most prominently the WNT/β-catenin pathway (also known as “canonical” WNT signaling). β-catenin is a key mediator of WNT signaling by acting as transcription factor to activate expression of WNT target genes. This WNT signaling pathway is a major regulator of many developmental processes, including axis specification, tissue patterning and organogenesis, and its deregulation during embryogenesis often has catastrophic consequences for the developing organism. In the adult, WNT signaling is critical for homeostasis of multiple tissues, including blood, intestine, skin and liver to name a few (reviewed in (Nusse, 2008). Inappropriate activation of WNT signaling has been observed in multiple cancers. A comprehensive analysis of human colorectal cancers found that over 90% of cancers harbor mutations in genes associated with WNT signaling (Cancer Genome Atlas, 2012). Deregulated WNT signaling has also been observed in cancers of the skin, liver, brain, breast and ovary.
The WNT signaling pathway was first elucidated in the Drosophila developmental mutant wingless (wg) and from the murine proto-oncogene int-1, now Wnt1 (Nusse & Varmus, 1982, Cell 31:99-109; Van Ooyen & Nusse, 1984, Cell 39:233-40; Cabrera et al., 1987, Cell 50:659-63; Rijsewijk et al., 1987, Cell 50:649-57). Wnt genes encode secreted lipid-modified glycoproteins of which 19 have been identified in mammals. These secreted ligands activate a receptor complex consisting of a Frizzled (“FZD”) receptor family member and low-density lipoprotein (“LDL”) receptor-related protein 5 or 6 (“LPR5/6”). The FZD receptors are seven transmembrane domain proteins with similarities to the G-protein coupled receptor (“GPCR”) superfamily and contain a large extracellular N-terminal ligand binding domain with 10 conserved cysteines, known as a cysteine-rich domain (“CRD”) or Fri domain. There are ten human FZD receptors: FZD1-10. Different FZD CRDs have different binding affinities for specific WNTs (Wu & Nusse, 2002, J. Biol. Chem. 277:41762-9), and FZD receptors have been grouped into those that activate the canonical β-catenin pathway and those that activate non-canonical pathways, all of which are described below (Miller et al., 1999, Oncogene 18:7860-72). To form the receptor complex that binds the FZD ligands, FZD receptors interact with LRP5/6, single pass transmembrane proteins with four extracellular EGF-like domains separated by six YWTD amino acid repeats (Johnson et al., 2004, J. Bone Mineral Res. 19:1749).
Three WNT signaling pathways have been characterized: (1) the canonical Wnt pathway; (2) the non-canonical planar cell polarity pathway; and (3) the non-canonical WNT/calcium pathway.
The canonical WNT signaling pathway activated upon receptor binding is mediated by the cytoplasmic protein Dishevelled (“DSH”) interacting directly with the FZD receptor and results in the cytoplasmic stabilization and accumulation of β-catenin. In the absence of a Wnt signal, β-catenin is localized to a cytoplasmic destruction complex that includes the tumor suppressor proteins adenomatous polyposis coli (“APC”) and Axin. These proteins function as critical scaffolds to allow glycogen synthase kinase (“GSK”)-3β to bind and phosphorylate β-catenin, marking it for degradation via the ubiquitin/proteasome pathway. Accumulated cytoplasmic β-catenin is then transported into the nucleus where it interacts with the DNA-binding proteins of the Tcf/Lef family to activate transcription.
In addition to the canonical signaling pathway, WNT ligands also activate β-catenin-independent pathways (Veeman et al., 2003, Dev. Cell 5:367-77). Non-canonical WNT signaling has been implicated in numerous processes, including gastrulation movements via a mechanism similar to the Drosophila planar cell polarity (“PCP”) pathway. Other potential mechanisms of non-canonical WNT signaling include calcium flux, JNK, and both small and heterotrimeric G-proteins. Antagonism is often observed between the canonical and non-canonical pathways, and some evidence indicates that non-canonical signaling can suppress cancer formation (Olson & Gibo, 1998, Exp. Cell Res. 241:134; Topol et al., 2003, J. Cell Biol. 162:899-908).
The canonical WNT signaling pathway also plays a central role in the maintenance of stem cell populations in the small intestine and colon, and the inappropriate activation of this pathway plays a prominent role in colorectal cancers (Reya & Clevers, 2005, Nature 434:843). Stem cells reside in the crypts of the absorptive epithelium of the intestines and slowly divide to produce rapidly proliferating cells that give rise to all the differentiated cell populations that move up out of the crypts to occupy the intestinal villi. The WNT signaling cascade plays a dominant role in controlling cell fates along the crypt-villi axis and is essential for the maintenance of the stem cell population. Disruption of WNT signaling, e.g., genetic loss of Tcf7/2 by homologous recombination (Korinek et al., 1998, Nat. Genet. 19:379) or overexpression of Dickkopf-1 (Dkk1), a potent secreted Wnt antagonist (Pinto et al., 2003, Genes Dev. 17:1709-13; Kuhnert et al., 2004, Proc. Nat'l. Acad. Sci. 101:266-71), results in depletion of intestinal stem cell populations.
All three pathways are activated by the binding of a WNT-protein ligand to a FZD. FZD7 is one of ten identified human WNT receptors.
FZD7 is expressed in the epiblast of the developing mouse embryo (Kemp C R, et al. Expression of Frizzled5, Frizzled7, and Frizzled10 during early mouse development and interactions with canonical Wnt signaling. Dev Dyn. 2007; 236(7):2011-2019) and that the human homolog FZD7 is elevated in undifferentiated human embryonic stem cells (“hESCs”) (Melchior K, et al. The WNT receptor FZD7 contributes to self-renewal signaling of human embryonic stem cells. Biol Chem. 2008; 389(7):897-903; and Sperger J M, et al. Gene expression patterns in human embryonic stem cells and human pluripotent germ cell tumors. Proc Natl Acad Sci USA. 2003; 100(23):13350-13355).
Indeed, FZD7 is specifically expressed in human pluripotent stem cells (“hPSCs”) (Fernandez et al., 2014, PNAS, 111(4):1409-14). The inventors previously demonstrated that FZD7 is abundantly expressed in hPSCs and, in fact, based on RNA-seq analysis, FZD7 is the most highly expressed FZD gene in hPSCs. Additionally, the inventors discovered that FZD7 expression declines upon differentiation, and knockdown of FZD7 expression using shRNAs disrupts the pluripotent state of hPSCs. Therefore, FZD7 is a stem cell specific WNT receptor. In addition, FZD7, along with FZD1, 5 and 8, is also expressed in mouse crypt epithelial preparations, which harbor intestinal stem cells (Hughes et al., 2011).
FZD7 expression has also been detected in several cancer cell lines and tumors, including breast cancer (Chakrabarti et al., 2014; Simmons et al., 2014; Yang et al., 2011), ovarian cancer (Asad et al., 2014), hepatocellular carcinoma (Merle et al., 2005; Nambotin et al., 2011; Nambotin et al., 2012; Song et al., 2014; Wei et al., 2011), Wilms' tumor (Pode-Shakked et al., 2011), gastric cancers (Kirikoshi et al., 2001) and colon cancer (Ueno et al., 2009; Vincan et al., 2010). Moreover, in view of the central role for FZD7 in stem cells (discovered by the inventors) and its limited expression in adult tissues, FZD7 may represent a unique cancer stem cell marker that can be targeted with minimal adverse side effects.
However, there are currently no methods to block signaling by specific FZD receptors. Several methods exist that block or inhibit WNT signaling, however, none of these methods block signaling through specific FZD receptors. For example, the following methods have been used to block WNT signaling: (1) extracellular proteins that bind WNT proteins, for example, recombinant SFRP and FZD-CRD-Fc fusions have been used to bind WNT and thereby block its ability to bind receptors; (2) extracellular proteins that block WNT/beta-catenin signaling, for example, recombinant proteins, such as Dkk1; (3) antibodies that bind to multiple FZD receptors, for example, Vantictumab (OMP-18R5, OncoMed Pharmaceuticals, Inc.), which is currently in clinical trial for the treatment of pancreatic cancer (ClinicalTrials.gov Identifier: NCT02005315), metastatic breast cancer (NCT01973309), and solid tumors (NCT01957007), binds to the extracellular domain of FZD7 and cross-reacts with 5 other FZD proteins, i.e., FZD1, 2, 5, 7 and 8 (Gurney et al. 2012); and (4) small molecule inhibitors.
However, none of these WNT inhibitors block the action of individual WNT ligands or FZD receptors. Therefore, although capable of blocking WNT-FZD7 signaling, these compounds interfere with other WNT signaling pathways as well, which may be essential for tissue and organ homeostasis. Given the large number of WNTs (19 human genes) and multiple FZD receptors (10 human genes), it is critical to disrupt signaling by specific WNT-FZD interactions without interfering with other WNT signaling pathways, which may be essential for tissue and organ homeostasis.
The present invention addresses the need for inhibitors that are specific to an individual WNT signaling pathway and provides novel antibodies and antibody fragments, e.g., Fabs, that specifically bind to human FZD7 without substantially interacting with (binding to) other FZD family members.